Acta Veterinaria et Zootechnica Sinica

The Signaling Mechanism of GDNF Expression in Sertoli Cells of Piglet Testis Regulated by FSH

SUN Yan;WANG Xianzhong;WU Jianyun;ZHU Fengwei;XIANG Yang;YANG Weirong;ZHANG Jiahua;

2011, 42(10): 1351-1356. doi:

Abstract ( 429 )

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This experiment was conducted to study the mechanism of FSH regulating GDNF expression in Sertoli cells. Different factors (inhibitor or activator) of signal pathway was added to Sertoli cells in vitro cutured, RTPCR and Western blot were used to detect the effect of FSH on GDNF expression. The result showed that: (1) FSH（50 ng·mL-1） could active ERK1/2 prominently along with a maximum stimulation at 2 h, the ERK1/2 reached a peak activity at 30 min. (2)dbcAMP（100 μmol·L-1）could not only increase the activity of ERK1/2 but also enhance the level of GDNF protein and mRNA with a maximum stimulation at 2 h. (3) While adding U0126 (10 μmol·L-1, MEK1/2 inhibitor) or H89 (10 μmol·L-1, PKA inhibitor) to culture medium, the results showed that the expression level of GDNF protein, mRNA and activity decreased apparently as compared with that in the cells treated with FSH alone. (4) The FSHactivated ERK1/2 in the cultured SC was timedependent, with a peak activity at 20 min. The activation of ERK1/2 by FSH was blocked by PKA inhibitor H89. These results indicate that FSH can activate ERK1/2 via cAMPPKA, which increase the expression of GDNF in Sertoli cell.

Study on Isolation, Culture and Multiple Differentiation Potential of Mongolia Horse Bone Marrow Derived Mesenchymal Stem Cells

ZHANG Yanru;LIU Zongzheng;WEI Lingai;SU Xiaohu;ZHAO Yiping;MANG Lai

2011, 42(10): 1357-1361. doi:

Abstract ( 401 )

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To establish a method for effective isolation and amplification of Mongolia horse bonemarrow derived mesenchymal stem cells (MSCs) and verification by morphology and multiple differentiation potential. MSCs were isolated from Mongolia horse bone marrow by gradient centrifugation and different adherent time method. Morphology and growth characteristics were examined by phase contrast microscopy. MSCs were induced in vitro to adipocytes by dexamethasone, IBMX, insulin and rosiglitazone, and to osteoblasts by βglycerophosphate, Lascorbicacid2phosphate and dexamethasone. The result showed that primary and passage cells were spindleshaped and presented active proliferation. Cells were successfully induced to adipocytes and osteoblasts cells, and presented the characteristics of adipocytes and osteoblasts cells. The result indicate that the method is established to isolate and multiply MSCs from Mongolia horse bone marrow effectively and to verify by morphology and its multipotential differentiation. It prove that the purified cells are not adult somatic cells but matrix cells.

Study of Inducing Rabbit BMSC into Cadiocyte

WANG Xinzhuang;HE Xiaoe;YAN Yingying;REN Weiqing;HE Jundan;LV Jingyu

2011, 42(10): 1362-1367. doi:

Abstract ( 429 )

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To investigate the method by which rabbit mesenchymal stem cells（MSCs）could be induced to cardiomyocytes and effect, MSCs isolated from rabbit femoral bone marrow were cultured in cell culture medium and in medium with 5Azacytidine（5Aza）. Whether the induced cells expressed cardiacspecific protein αactin and cardiac troponin T（cTnT） was identified by immunocytochemistry and inverted microscope, and the differentiation rate in each group was calculated. The results showed that some MSCs gradually increased in size and formed a sticklike appearance after inducation in experimental group, one month after treatment, most of the long sticklike cells connected with adjoining cells and began to form myotubelike structures, the direction of the cells arraying was similar gradually. Cardiac cellspecific protein αactin and cTnT expressed positively, the expression rate of cardiac cellspecific protein was different after MSCs was induced by different concentration 5Aza, of which 10 μmol·L-1 5Aza was the optimal concentration. These results indicate that MSCs can be induced to cardiomyocytes by 5Aza in vitro, and which have the characteristics of cardiomyocytes in structure. The optimal concentration of 5Aza inducing MSCs to cardiomyocytes is 10 μmol·L-1.

Establishment of Sheep Fibroblast Cell Line with Myostatin Gene Silencing

TANG Dayun;;ZHU Huabin;WU Jianmin;DU Weihua;WANG Dong;ZHAO Xueming;CHEN Hanzhong;LIN Xiukun

2011, 42(10): 1368-1373. doi:

Abstract ( 425 )

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The aim of this study were to obtain fibroblast cell line with Myostatin silencing, to further understand the regulation mechanism of Myostatin and to get Myostatin silencing transgenic sheep. Four siRNAs were designed according to Myostatin gene sequences and Myostatin silencing pSilencer vectors were constructed, and then transfected into fibroblasts, the silencing effects were analysised by RTPCR; A lentiviral vector with Myostatin gene silencing was constructed and then lentivirus particles was packaged. Fibroblasts were infected by lentivirus particles and positive cells were isolated by flow cytometry. Positive cells were identified by sequencing and the silencing effect was analyzed by RTPCR. An effective siRNA sequence(PSL1) was obtained, and it inhibited the Myostatin expression significantly with a rate of 90%. The virus particles which were 108 titer determined by fluorescence microscope were obtained. Positive cell line with the purity of 99% were isolated by flow cytometry. The result indicate that positive transgene cell line without resistance marker can be quickly obtained by using lentivirus and flow cytometry. This study provides a useful cell line for further research the regulation mechanism of Myostatin and get transgenic sheep.

Construction of Lysostaphin Mammaryspecific Expression Vector and Transfection of Somatic Cells to Provide Transgenic Donor Cell for SCNT

SHANGGUAN Tao;ZHANG Bowei;SUN Weiwei;HUANG Xin;ZHANG Yong;

2011, 42(10): 1374-1379. doi:

Abstract ( 407 )

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The purpose of this study was to prepare lysostaphin transgenic donor cells for somatic cell nuclear transfer by constructing mammaryspecific expression vector and transfecting bovine fetal fibroblasts. In this study，pEGFPC1 was used as the vector backbone, 2.6 kb 5′ regulatory region of and 0.6 kb 3′ flanking region of bovine βcasein were used as regulatory regions, the mammary glandspecific expression vector pEPB was constructed, which included neomycin resistant gene and EGFP reporter gene. After identified by PCR and restrictive enzyme digestion, the pEPB was transfected into the bovine mammary epithelial cells through FuGene HD for 35 times, the transgenic cells were detected by immunofluorescence analysis after induced by prolactin（1 mg·mL-1）. And then, the plasmid pEPB was transfected into the bovine fetal fibroblast cells by electroporation, positive cells were screened by G418 selection and determined by PCR and Southern blot. The results showed that the lysostaphin gene was expressed in bovine mammary gland epithelial cells and integrated into bovine fetal fibroblasts genome. The results indicate that the lysostaphin transgenic fibroblast cells obtained may be competent for bovine transgenic cloning.

Study on Genetic Variation in Tibetan Yak by RAPD Technique

CHAI Zhixin;ZHAO Shangjuan;JI Qiumei;ZHANG Chengfu;XIN Jinwei;ZHONG Jincheng

2011, 42(10): 1380-1386. doi:

Abstract ( 392 )

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To understand the genetic diversity of Yak populations from Tibetan and the relationship among them, a total of 33 polymorphic primers were used in RAPD analysis in 11 populations including Baqing Yak , Leiwuqi Yak, Dingqing Yak, Sangri Yak, Gongbujiangda Yak, Jiangda Yak, Kangbu Yak, Sangsang Yak, Jiali Yak, Pali Yak, Sibu Yak， 8 of them were selected for RAPD analysis of DNA of 11 Yak populations. Genetic distance indexes among breeds and genetic similarity indexes within breeds were calculated by Nei and the phylogenetic tree was constructed by UPGMA cluster analysis. The result showed that genetic diversity index of Tibet yak breeds or groups varied between 0.185 7 and 0.405 3. Among them Pali Yak was 0.185 7, which indicated that Pali Yak was relatively pure, the group was neat. And Gongbujiangda Yak was 0.405 3, which displayed that group internal have more variations. In 11 breeds or groups, the genetic diversity index respectively were: Gongbujiangda Yak(0.405 3)＞Jiangda Yak (0.353 6) ＞Sibu Yak(0.344 8)＞Kangbu Yak(0.342 8)＞Jiali Yak(0.332 3)＞Sangri Yak(0.282 3)＞Baqing Yak(0.279 3)＞Sangsang(0.269 8)＞Dingqing Yak(0.259 7)＞Leiwuqi Yak(0.224 1)＞Pali Yak(0.185 7). The eastern Tibet yak breeds or groups had higher genetic diversity, and the western Tibet yak had lower genetic diversity, which indicated that eastern Tibet may be the origin of yak. The molecular clustering relationship chart showed that Tibet 11 yak breeds or groups could be divided into two clusters. Pali Yak was a cluster, the others were another cluster. The result indicate that Tibet yak have abundant genetic diversity and significant genetic difference among the breeds or groups.

Effect of Dietary Protein on the Growth Performance and the Regularity of Digestibility and Metabolism in Mink at the Period of Development

ZHANG Tietao;ZHANG Zhiqiang;REN Erjun;GAO Xiuhua;YANG Fuhe;XING Xiumei

2011, 42(10): 1387-1395. doi:

Abstract ( 504 )

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The expriment was designed to investigate the regulatory of digestion and metabolism of different dietary protein levels in mink at the period of the growth and development. Sixty young male minks were randomly divided into six groups according to a singlefactor test. The trials of feeding and metabolism were used to analyse the digestibility of protein, digestibility of fat, nitrogen deposition and protein biological value, combining with the ratio of feed conversion rate and the average daily gain to deduce the fluctuation regulation of nutrition in mink during the rearing process of the growth and development. The result showed that: the paremeters such as body weight, protein biological value and net protein utilization were significantly different among groups(P<0.05); feed conversion rate, digestibility of dry matters, digestibility of protein, digestibility of crude fat, intake nitrogen and urine nitrogen were greatly significantly different among minks (P<0.01); the more urine nitrogen was drained with the more dietary protein (P<0.05); the percentage of dietary protein was less than 32%, the feed conversion rate and average daily gain of minks were significantly reduced(P<0.05). The content of dietary protein was lower than 32%, the growth rate of mink would be obviously declined(P<0.05); the higher dietary protein level that over 34% promoted the digestibility of crude fat and dry matters;the optimum dietary protein level were 36% and 34% at 5080 day ages and 80110 day ages, respectively.

Construction and Coexpression of Bicistronic Expression Vector Based on FMDV 2A in Mammary Gland Cells

CHEN Qiuju;WANG Yunfei;CUI Yihong;ZHU Guangqin;YANG Mingming;SONG Yuxuan;CAO Binyun

2011, 42(10): 1396-1401. doi:

Abstract ( 437 )

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The aim of this study was to construct mammary gland specific bicistronic expression vector based on FMDV 2A, and verify the expression of sequences of EGFP and human IL2 in mammary epithelial cells. We utilized selfcleaving peptide 2A from footandmouth disease virus (FMDV 2A) connecting hIL2 and EGFP sequence, and cloned into goat βcasein regulatory sequence, then we got mammary gland specific bicistronic expression vector pFIENβ, after transient transfection into goat mammary epithelial cells by LipofectamineTM 2000, we used RTPCR and Western blot to detect the expression of hIL2 and EGFP. Restriction analysis suggested that the recombinant plasmid pFIENβ is correct, then we found that the cells which were transfected with pFIENβ and pEGFPC1 expressed EGFP by observing with fluorescent microscope, and there is no green flourescence in which were untransfected, and we amplificating hIL2 and EGFP sequence in the cells transfected with pFIENβ, and amplificating EGFP sequence only in the cells transfected with pEGFPC1; After detection by Western blot, the cells transfected with pFIENβ expressed both hIL2 and EGFP. The results showed that goat βcasein regulatory sequence can trigger hIL2 and EGFP expression in mammary epithelial cells, and FMDV 2A facilitate expresson of hIL2 and EGFP independently.

Isolation and Biological Characterization of Canine Parvovirus Isolates

ZHOU Yunduo;KANG Zhenyu;CHEN Yueping;SHI Xiaona; CHEN Jianguo;ZHAO Yaxin;LIU Zhengfei

2011, 42(10): 1402-1408. doi:

Abstract ( 407 )

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The aim of this study was to obtain Canine Parvovirus (CPV) strains in the middle of China and study their genetype, biological characterization and pathogenicity. 14 feces swabs that were positive by colloidal gold test were collected from pet clinics in Wuhan. Then they were passaged in Feline kidney 81 (F81). 2 pairs primers were designed according to conservative Capsid protein 2 (VP2). The strains were purified by plaque assay, and then onestep growth curves were obtained. Subsequently transmission electron microscopic (TEN) sections were made. And the animal experiment was carried out. 5 CPV strains were isolated. 390 bp and 583 bp nucleotide sequences were acquired. After sequencing and analyzing the gene fragments, the result shows that 4 strains were CPV2a and 1 was CPV2b. The curves show the growth of CPV at different stages after infection. The reproductive activity can reach or beyond 105.5 PFU·mL-1. The photographs of TEN showed that mitochondria are swelling. Finally, animal experiment shows that every dog had clinical symptoms in different degrees. The results of dissection reflect that there are necrosis in the small intestine and the myocardium becomes thinner. After pathological sections were made, observation was taken up and the result shows that myocardial cells are metamorphic and necrotic. This study provides groundwork for further study of molecular biology and pathopoiesia of CPV.

Detection of Pathogenic Aeromonas hydrophila by Dot-ELISA

REN Yan;PAN Zihao;LU Chengping;YAO Huochun; WU Shuqin

2011, 42(10): 1409-1415. doi:

Abstract ( 397 )

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On the basis of diagnosis kit for pathogenic Aeromonas, ECPase54 produced by Aeromonas hydrophila (Ah) was detected by DotELISA. On the other hand, the 16S rRNA and aerolysin of pathogenic Aeromonas hydrophila (PAh) were detected with PCR. Furthermore the skimmed milk plate tests were processed. 72 Aeromonas isolates were detected with the 4 different methods. The positive rate of 72 isolates was 90.3%（65/72）with DotELISA method , 75%（54/72）with skimmed milk plate method, 94.4%（68/72）with aer gene PCR method, 81.9%（59/72）with 16S rRNA PCR method. Compared DotELISA and the other three methods(skimmed milk plate, aer gene PCR and 16S rRNA PCR), the coincidence rate was 79.2%（57/72）, 91.6%（66/72）, 81.9%（59/72），respectively. The 72 isolates were repeatedly detected with DotELISA in 2, 4, 6, 12 and 18 months after preparing of the antiECPase54 rabbit serum, and the similarly results were got. In conclusion, this DotELISA method is sensitive, specific and practical. It can be used as a method for rapid diagnosis of PAh in clinic.

The Current Status of the Drug Resistance and Evolutionary Relationship of MSSA and MRSA Isolates from Bovine of China

WANG Dengfeng;DUAN Xinhua;WU Jianyong;YANG Xueyun;LI Jianjun;LI Na;WANG Zhicai

2011, 42(10): 1416-1425. doi:

Abstract ( 442 )

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The aim of this study was to investigate the current status of the antimicrobial resistance of bovine Staphylococcus aureus isolates, so as to do research on carried drugresistant genes and molecular epidemiology profiles of methicillinsusceptibility Staphylococcus aureus（MSSA）and methicillinresistant Staphylococcus aureus（MRSA）which isolated from bovine, further, to reveal the evolutionary relationship between MSSA and MRSA, and origin and diffusion of MRSA. Total 54 isolates which isolated from Xinjiang, Zhejiang, Shandong, Neimenggu and Shanghai in 2009 were investigated antibiotics susceptibility by disc diffusion method, and 12 isolates of MSSA and MRSA which confirmed by Cefoxitin sensitive test by disc diffusion method and mecA PCR were used to detected the antimicrobial resistance genes and typed by multilocus sequence typing (MLST). Drug sensitive test results showed that 88.8% isolates resisted to erythromycin, 81.5% to clindamycin, 88.9% to penicillin, 90.7% to ulfamethoxazole compound, 92.6% to doxycycline, 94.4% to tetracycline, 79.6% to chloramphenicol, 63.0% to ciprofloxacin, 70.4% to gentamicin. Besides that, 5.6% isolates resisted to all 10 antibiotics, 85.2% isolates resisted to more than 5 antibiotics, 6 isolates (11.1%) resisted to cefoxitin. Furthermore, 3 MRSA isolates resisted to 10 antibiotics, Xinjiang MSSA isolates resisted to 24 antibiotics, other provinces MSSA isolates resisted to 7 antibiotics, and analyzed result of the antimicrobial resistance gene of MSSA and MRSA, found all isolates carried ermC and tetK genes, but no ermA gene, 6 MRSA isolates carried aac(6′)/aph(2″), 5 and 4 MRSA isolates carried tetM and aph(3′) Ⅲ respectively, but only 4 MSSA isolates carried tetM gene, no MSSA isolates carried aac(6′)/aph(2″) and aph(3′)Ⅲ. Staphylococcal chromosomal cassette (SCC) mec of MRSA was typed by multiPCR, the results showed that 5 isolates were SCCmec IV and 1 isolate can not identificated type. The MLST results of MSSA and MRSA proved that the isolates belonged to ST50, ST965，ST6，ST97 and ST9, further analysis found that 6 MRSA isolates distributed in CC7，CC4，CC21 and CC9 and MSSA isolates distributed in CC32 and CC7. The results suggest that drug resistance of Chinese bovine Staphylococcus aureus isolates are serious and most isolates represent multidrug resistance, MSSA and MRSA are multidrugresistant strains and carries more than 2 antimicrobial resistance genes, epidemical bovine MRSA is MRSASCCmec IV. By analyzing the results of MLST and comparing with MLST results of human MRSA, found that, in China, bovine MRSA was different with human’s and speculated that bovine MRSA emerged when MSSA which belonged to different CCs gained SCCmec IV by antibiotic selection, in addition, MRSA stains isolated from different areas were not the same ancestor, further analysis, presume those genes similar to plasmid or transposon can free communication in different isolates than depend on SCCmec.

Effect of Newcastle Disease Viruses on Morphology and the GABA Expression of Hypothalamus-pituitary-adrenocortical Axis in Ostrich Chicks

TANG Li;WEI Lan;PENG Kemei;CHEN Zhengli;FANG Jing;DENG Tianhuai;PAN Kangcheng;HE Min

2011, 42(10): 1426-1431. doi:

Abstract ( 421 )

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New castle disease (ND) is one of the most serious infection diseases that damage ostrich cultivation. As a stressor, ND could invade the organism and cause a series of pathological changes in body, which induced excessive activation or inactivation of ostrich chicks' hypothalamuspituitaryadrenocortical axis (HPA), and seriously disturbs the organismic internal environment. In the present study, ostrich chicks aged 45 days were divided into normal group and challenge group at random. The methods of HE staining, transmission electron microscopy (TEM)，and immunohistochemistry (SABC) were used to study the changes of morphology and theγaminobutyricacid (GABA) expression in ostrich chicks’ HPA axis with challenge by Newcastle disease virus（NDV）. The results are as follows: The ostrich was highly sensitive with NDV, which can cause significant change on histopathology of HPA axis. The adrenalopathy worsened seriously, followed by hypothalamus, and a pituitary disease was a little lighter, which reflected the damage degree of the HPA. (2) Infected with NDV, the GABA expression of HPA axis in ostrich downgraded, change regularity in adrenals shows that GABA may participate in stress reaction of HPA axis which was caused by ND. The expression in the number and intensity of is related to pathological stress reaction.

The Changes of marA，soxS and robA Gene Expression Levels in Three Kinds of Antimicrobial Agents Induced Resistant Mutants in vitro

LIU Jianhua;YUAN Li;PAN Yushan;CHEN Yuxia;HU Gongzheng;WU Hua;GUO Fanxi

2011, 42(10): 1432-1437. doi:

Abstract ( 385 )

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The research was conducted to investigate the changes characteristics of the expression level of active efflux system regulatory genes (marA, soxS and robA) in Escherichia coli O78which induced by different antimicrobial agents. Extracted the total RNA of the 10th, 20th, 30th generations of ceftriaxone induced resistant mutants, ciprofloxacin induced resistant mutants and amikacin induced resistant mutants respectively. The mRNA expression levels of active efflux system regulatory genes, such as marA, soxS and robA were determined by reverse transcription realtime fluorescence quantitative PCR. The result showed that the mRNA expression level of marA gene in CIP20 was the highest, it was 2.30 times compared with the parent strain, and in CIP10, CRO30, CIP30 , AMK20 were 2.11, 1.71, 1.15, 1.44 respectively; The soxS gene expression level in AMK20, CRO30, CRO20, CIP20 and CIP30 were higher than O78, AMK20 was the highest one, which was 5.98 times of O78; Concerning the expression of robA genes, except CIP10 was increasing, all the other strains was decreased when compared with O78. The results indicated that ceftriaxone sodium, ciprofloxacin and amikacin can induce the expression of marA, soxS gene increased, robA gene decreased.

Preparation of Doxycycline Hyclate Microcapsule and Its Pharmacokinetic in Rabbit

FU Hualin;ZHANG Li;ZHANG Wei

2011, 42(10): 1438-1444. doi:

Abstract ( 393 )

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The objective of this study was to prepare the chitosanmicrocapsule of doxycycline hyclate by emulsifying crosslinking method, and evaluate its surface morphology, particle size, drug encapsulation efficiency, in vitrorelease et al. Doxycycline hyclate solution and microcapsule suspension were orally administrated on health rabbits to study the pharmacokinetic of doxycycline hyclate microcapsule. Six rabbits received single doses of doxycycline hyclate solution at 20 mg·kg-1, and the other six rabbits received doxycycline hyclate suspension at the same doses. The plasma concentration of doxycycline hyclate was determined by HPLC. The stationary phase was C18, and acetonitrilemethanol0.01 mol·L-1 oxalic acid solution (2:1:7) was taken as the mobile phase, AUV detector was used at 346 nm. Using doxycycline hyclate solution as a reference, their pharmacokinetic differences were compared. The results indicated that the microcapsule was successfully prepared. SEM observation showed the drugloaded microparticles exhibited spherelike shape and small particle size was about 10 μm. The mean content and encapsulation efficiency were 20.60% and 85.54%, respectively; In vitro release studies revealed that the drugloaded microparticles substantially enhanced the release property. The pharmacokinetic data of two groups was in line with a twocompartment model. The main pharmacokinetic parameters were shown as Cmax of (1.74±0.00) vs. (1.10±0.00) mg·L-1, Tmax of 3 vs.12 h, T1/2α of (1.16±0.53) vs. (7.51±2.87) h，T1/2β of (2.19±0.38) vs. (9.00±1.60) h，and AUC(0t) of (11.71±0.17) vs. (32.51±0.20) mg·L-1·h, respectively. The relative bioavailability of the microcapsule was 289.4%. It resulted that doxycycline hyclate microcapsule exhibited high bioavailability and was slowly absorbed and distributed after administration in the healthy rabbit.

The Morphological Structure of Bone Cavity Hole of Skull in Jianghan Buffalo

JIN Wei;JIN Shengzao;WANG Jiaxiang;LV Fubin

2011, 42(10): 1445-1449. doi:

Abstract ( 435 )

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The buffalo is one of the most important animals to employ as a servant and animal food of modern countryside in our country. So many researches on the buffalo have been performed and the anatomical investigation with buffalo is one of the basic researches. To provide more basic data for the further research on buffalo, we have investigated on the morphological structure of bone cavity hole of skull in Jianghan buffalo in this study. The shape and position for every bone hole in skull cavity for the Jianghan buffalo were dissected and surveyed, and the corresponding nerves, blood vessels and other organization distribution in the bone hole were also investigated. The diameters with every bone hole were measured by measurement method. The results showed that there were total 13 bone holes in the skull of Jianghan buffalo, and it was existed the corresponding nerves and blood vessels in every bone holes. The diameter for each bone hole as follows: supraorbital foramen was 6.74 mm, suborbital foramen was 6.64 mm, inferior foramen occipital was 45.42 mm, ala temporalis orbital hole was 5.33 mm, the hole under palate was 7. 85 mm, wing foramen ovale was 10.73 mm, temporal joint deuterostoma was 4.47 mm, jugular foramen was 9.87 mm, foramen hypoglossi was 9.05 mm, temporal canal hole was 14.46 mm, mandibular foramen was 6.28 mm, mental foramen was 4.21 mm and cribriform foramina was 3.37 mm. This work of centralized investigation on the morphological structure of bone cavity hole of skull and the corresponding nerve, blood vessels and other organization distribution for Jianghan buffalo is to provide some scientific bases with the anatomical investigation and teaching for Jianghan buffalo.

Micromorphological Characteristics of the Adult Tibetan Plateau Sheep Spermatic Cord

YUAN Ligang;SUN Ying;HUANG Bumin

2011, 42(10): 1450-1456. doi:

Abstract ( 379 )

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The Tibetan Plateau sheep is one of the ordinary domestic animal of QinghaiTibetan Plateau, while anatomical evidence of its special testicular microvasculature adaptability to the high altitude environment is still less. By using the methods of vascular proplasm and replica scanning electron microscopic, we studied the angioarchitecture of the Tibetan Plateau sheep spermatic cord and its functional relationship. The results were as follows: 1)The convoluted testicular artery, which was embedded in the network of the venous pampiniform plexus and gave off branches to supply the epididymis; 2)The collecting veins, which run parallelly and anastomose frequently and formed a “Binding Area of Pocket”, it was the initiation area of the pampiniform plexus; 3)The testicular artery and pampinifotm plexus could be divided into three grades correspondingly, the vasa vasorum deriving from branches of testicular artery to supply the wall of pampiniform plexus. From the above, the specific angioarchitectural characteristics of spermatic cord in Tibetan Plateau sheep are very different with other adult mammals.

Acetamiprid Impairs the Ultrastructure of Testes Via Inducing Oxidative Stress in Mice

ZHANG Jiaojiao;WANG Yi;XIANG Haiyang;WANG Xianzhong;ZHANG Jiahua

2011, 42(10): 1457-1462. doi:

Abstract ( 382 )

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The objective of this study was to explore whether the impairment of acetamiprid on the ultrastructure of sertoli cells and spermatogenic cells was associated with oxidative stress. Fifty adult Kunming male mice (2530 g) were divided into five groups (n=10 per group). All groups were treated for 35 days by gavage. The results showed that, in the acetamiprid alone group, the endoplasmic reticulum was expanded in sertoli cells, and there were numerous lysosomes. The nucleus of primary spermatocytes was dissolved and mitochondrial was pyknosised; chromatin aggregated abnormally. Furthermore, the nuclear chromatin of spermatids was aggregated abnormally, and emerged apparent chromatinlike corpuscles, while mitochondrial was pyknosised significantly. Acetamiprid increased the concentrations of total NO synthase (TNOS) and induce NO synthase (iNOS) in testes and the capability of producing hydroxyl radical (P<0.05, for all), but reduced the activity of glutathione (GSH) and total antioxygen capability (TAOC) (P<0.05, for both). Vitamin E significantly ameliorated the impairment of acetamiprid on the ultrastructure of sertoli cells and spermatogenic cells. Therefore, acetamiprid impairs the ultrastructure of testes via inducing oxidative stress in male mice.

Effect of Heat Shock on HSP 70 Expression and Ultrastructure of Mouse Embryonic Cell

ZHANG Xia;QU Pingping;CONG Xia;JIANG Zhongling;CUI Kai;TIAN Wenru

2011, 42(10): 1463-1469. doi:

Abstract ( 403 )

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The aims of this study were to determine the developmental stage when HSP 70 was synthesized in the mouse embryo induced by heat shock in vitro and to study the effect of heat shock on the ultrastructural changes of the embryo in the corresponding period. The zygotes and embryoes at the 2cell, 4cell, 816cell and blastular stages were treated with heat stress at the temperature of 37, 39 and 41 ℃, respectively, to determine the expression of HSP70 by the method of immunofluorescence and cytochemistry. The mouse embryos at the early blastular stage were treated separately in vitro at 39 and 41 ℃ for 2 h. Then the embryos were collected at 0 and 2 h after heat shock to make ultrathin section; the transmission electronic microscope was used to observe the change of embryo ultrastructure. The expression of HSP70 was different at different embryonic development stages. There was a negative (-) expression in the 37 ℃ group of all stage embryo. However, in the 39 ℃ group of embryos, there was a weak expression (+) of HSP70 in the 816cell stage and a positive expression (++) at the blastular stage. In the 41 ℃ group, HSP70 was weakly expressed (+) at the 4cell stages and positively expressed (++) both at 816cell and blastular stages. There was a significant change in the ultrastructure of early blastula induced by both 39 and 41 ℃ heat shock compared to the control group. The ultrastructural change of embryo organelle treated in 39 ℃ group recovered to normal by culture at 37 ℃ for 2 h. However, the recovery was not occurred in the embryos treated at 41 ℃. These results indicate that the HSP70 is induced to synthesize in the embryos from 4cell stage and is mostly synthesized at the blastular stage; heat shock changes subcellular structure of the embryo, which appear to have degenerative changes, however, the mild changes are reversible.

Studies on the Regulation of Egg Laying and Incubation in Hens after Immunization against Vasoactive Intestinal Polypeptide

LIU Ying;CHEN Feng;HUANG Yunmao;LIANG Shaodong;SHI Zhendan

2011, 42(10): 1470-1476. doi:

Abstract ( 358 )

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This study was conducted to investigate the regulatory mechanisms of egg laying and development of incubation behaviour by immunization against vasoactive intestinal peptide (VIP) in hens. In experiment 1, 18wk old prelay Taihe pullets were immunized on d1, d28, d56 and d84 of the experiment, with VIPBSA conjugate immunogen. In experiment 2, laying 23wk old Yuehuang hens were immunized on d1, d23, d48, d73 and d98 of the experiment, with tethered 4VIP recombinant protein as immunogen. Tissues of hypothalamus, pituitary gland, theca and granulosa layers of F1 to F3 ovarian follicles were collected, in 10 immunized and 10 control hens after slaughter on d98. The results of experiment 1 showed that immunization against VIP significantly attenuat the prepeak egg laying during d55 to d61 of the experiment 1, but significantly slowed postpeak decline of egg laying from d89 to d103 of the experiment. Meanwhile immunization against VIP also delayed the development of incubation behaviour. In experiment 2, the initial egg laying rate within 30 days after the experiment was low in both VIPimmunized and control hens, but was significantly higher in the former than that in the latter. Following increase of photoperiod from 12 h to 16 h, laying rate increased to the peak in both groups of hens. However VIPimmunized hens exhibited a substantial decrease in egg laying, which was significantly lower than that in the control group from d80 to d105 of the experiment. Percentage of hens incubating increased at a slower rate in the VIPimmunized hens than that in the control group, but were similar between the two groups at the end of the experiment. On d98 of the experiment, both groups of hens had similar numbers of large yellow, small yellow and large white follicles on the ovary. Immunization against VIP did not affect the mRNA expression level of GnRHⅠ, VIP in the hyothalamus, nor affected LHβ and FSHβ in the pituitary gland. However, PRLRmRNA expression level in both hypothalamus and pituitary, and PRL expression level in the pituitary gland were all significantly lower in VIPimmunized hens. In addition, immunization against VIP decreased PRLR mRNA expression in granulosa cells of F1 and F3 follicles, and decreased LHR mRNA level in granulose and theca layers of F1F3 follicles examined. Results of the 2 experiments demonstrated that immunization against VIP decreased gene expression and therefore secretion of PRL in the pituitary gland, which subsequently slowed development of incubation occuring after egg laying. Suppression of PRL secretion following VIPimmunization also depresses egg laying at certain stages, which may be a result of decreased LHR mRNA expression in the ovarian follicular cells.

Identification of BroodinessRelated Geese Genes by Suppression Subtractive Hybridization

GUO Jun;TANG Qingping;ZHANG Shuangjie;MA Yuehui;LU Huolin;SU Jiandong;ZOU Jianmin;CHEN Kuanwei;LI Huifang

2011, 42(10): 1477-1484. doi:

Abstract ( 394 )

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The aim of this study was to construct broodiness and laying geese cDNA libraries with SSH, and to identify differentially expressed genes between laying and broody phases. Ovary in broody and laying phases was ascribed to tester and driver for reciprocal SSH procedures. cDNA libraries of specifically or highly expressed genes in broodiness and laying were obtained after twostep hybridization. Positive clones from libraries were randomly selected to validate with PCR. Sequently, 654 positive clones were selected to sequence, qualify, cluster, assemble and compare in GenBank using BLASTn. 166 differentially expressed gene fragments from two libraries showed homology to other known sequences, including 92 functional genes. The expression profiles showed some broodinessspecified genes invovled in apoptosis, cell component, hormonesrelated signal transduction, and some genes involved in metabolism and cell defense in the layingspecified genes library; some ESTs belonged to unknown or hypothetical protein. The result indicate that three genes(prolactin receptor, estrogen receptor α and antimullerian hormone receptor II) in broodinessspecified library are associated to trigger or maintain broodiness in geese based on biological function and signal transduction. The result provide the basis for future revealing the mechanism on broody behavior.

The Development and Application of a Multiplex Reverse Transcription Polymerase Chain Reaction for Detection of Porcine Reproductive and Respiratory Syndrome Virus

WU Jinyan;TIAN Hong;SHANG Youjun;CHEN Yan;WEI Yanquan;HOU Xiangmin;ZHAO Na;HE Jianhui;LIU Xiangtao

2011, 42(10): 1485-1490. doi:

Abstract ( 440 )

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A multiplex reverse transcription polymerase chain reaction (multiplex RTPCR) was developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Three pairs of primer selected from four pairs were designed based on the sequence of high conservative region of PRRSV. And PCR product was 228, 345 and 490 bp. Diagnostic accuracy of the multiplex RTPCR shown to be 10fold more sensitive than the conventional single RTPCR (conventional RTPCR) method, and the testing time is shorten greatly. Simultaneously, the specificity and sensitivity of this method were evaluated. The result indicated that this assay could reliably differentiate between PRRSV and other swine viral disease, such as classical swine fever virus (CSFV), porcine rabies virus (PRV). The developed multiplex RTPCR in this study may provide a new avenue to the rapid detection of this important pathogen in one reaction, which possess the good character in specificity, sensibility，simple and convenient.

Mutipal Tumors Induced by Avian Leukosis Virus Subgroup J in Layer Flock

CHEN Hongbo;YU Linlin;JIANG Yanping;WANG Yue;WANG Feng;WANG Xiaowei;WANG Guihua;CHENG Ziqiang;

2011, 42(10): 1491-1496. doi:

Abstract ( 435 )

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In August 2009, low produce peak was observed by farm owner in Hyline layer chicken flock at Tai′an in Shandong province. The rate of produce only was 60%70%, and the mortality was up to 20% at age of 31week. The ill chickens were diagnosed as avian leukosis virus subgroup J infection by gross observation, pathology, PCR and immunohistochemistry. Except myelocytomas, fibrosarcomas, histiocytic sarcomas, intestinal adenocarcinoma and hemangioendothelioma were also observed in same or different organs. Fibrosarcomas exist single in ill chicken, and the other tumors were concurrent with myelocytomas. The histopathology detail characters of tumors were described in this paper. The etiology and mechanism of transmmition and tumor spectrum expended of ALVJ need to be further studied.

Construction and Identification of Support Plasmids and Minigenome of a Peste des Petits Ruminants Virus Isolate, China/Tib/07

LIU Wenhua;;WANG Zhiliang;BAO Jingyue;WU Xiaodong;LIU Hualei;ZHAO Yonggang;LIU Xiufan

2011, 42(10): 1497-1502. doi:

Abstract ( 425 )

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The research was conducted to construct and identify the support plasmids and minigenome of Peste des petits ruminants virus (PPRV) in order to lay a foundation for the establishment of a reverse genetic system for rescue of PPRV. Support plasmids of pCIN, pCIP and pCIL according to the PPRV isolate, China/Tib/07, were constructed and functions were identified by IFA and RTPCR after transfection. At the same time, the two end sequences of the genome of the PPRV isolate, along with the reporter gene of eGFP were amplified, then the 3 fragments were connected by overlapPCR and cloned into the transcription vector, pOLTV5, the minigenome was constructed and then cotransfected with the support virus and the 3 support plasmids respectively. As a result, the 3 support plasmids were identified to be correct and the reporter gene of eGFP expressed when the minigenome transfected with either the support virus or the 3 support plasmids. In conclusion, support plasmids of the N, P and L genes and minigenome of the PPRV were constructed successfully. Furthermore, the 3 plasmids worked well with the minigenome, which indicated that the 3 plasmids can work as the support plasmids for the study of reverse genetic operations of China/Tib/07.

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